Lentivirus-mediated gene transfer is a promising new approach for delivering genetic information to target cells to combat human disease, including diseases of the airway such as cystic fibrosis. However, a number of barriers, both physical and technical, must be overcome in order to implement this new technology safely and with high efficacy. This proposal, in three specific aims, outlines experiments designed to overcome physical barriers to successful transduction of well-differentiated human airway epithelia and also to improve the technical aspect of producing self-inactivating (SIN) lentiviral vectors by lentiviral packaging cells. The first specific aim describes novel approaches to vector pseudotyping in which the envelope proteins from human influenza A virus or human parainfluenza virus-3 are used to target lentiviral vectors to the apical membrane of polarized human epithelial cells. In the second specific aim, experiments are designed to characterize and overcome intracellular barriers that limit vector gene expression. The third specific aim describes the development of a double-SIN piggy-back vector that will improve the generation of packaging cell lines producing lentiviral vectors.